Column cleaning and regeneration

 As we all know, the column efficiency and peak shape of a chromatographic column will deteriorate after being used for a long time. So if the chromatographic column is contaminated, how should it be flushed or regenerated?

When cleaning the chromatographic column, generally use more than 20 times the column volume of each solvent to flush the chromatographic column. First, make sure there is no buffer in the chromatographic column, and the residual solvent in the chromatographic column before cleaning should be miscible with the latter cleaning solvent.

Corresponding flushing solutions for various types of pollution:

1. Salting out in reverse phase: flush with 95% water and acetonitrile at a low flow rate until the column pressure drops.

2. Strong adsorption of non-polar compounds in reverse phase: In the absence of salt in the column, ethanol washing followed by dichloromethane washing can basically wash away non-polar compounds.

3. Residual proteins in reverse phase: ① After transition with 95% water-acetonitrile, use 0.1% TFA water gradient elution to 0.1% TFA (acetonitrile/isopropanol 1:2); If it is an alkali-resistant chromatographic column, you can use 95% water-acetonitrile transition on the basis of ①, and then wash with 5M urea or 5M guanidine thiocyanate (pH7).

4. Residual metal ions in reverse phase: 0.05M EDTA.

5. Ion pair reagent contamination in reverse phase: 95% water acetonitrile transition, then rinse with 95% acetonitrile water. However, it is difficult to completely rinse the ion pair reagent after it is contaminated.

6. Protein residue in ion exchange or SEC: 5 M urea or 5 M guanidine thiocyanate (pH 7).

Tips: ① The replacement solvents before and after flushing should be miscible; ② Do not connect the detector when flushing the chromatographic column.